The proposed research involves comparative studies using both FIV and HIV to investigate drug resistance development and substrate specificity, defining the time-course and structural basis for resistance development with the ultimate goal of providing structural information relevant to developing broad-based inhibitors. FIVs encoding chimeric protease (PR) with mutations that impart drug sensitivities and substrate specificities similar to HIV-1 PR will be generated for analysis against a panel of specific peptide substrates and phage libraries and for ex vivo infections. Progress of infections will be monitored to examine the molecular course of drug sensitivity/resistance development under single and multiple drug selections. Cleavage of peptide substrates for HIV by chimeric PRs will be used to identify residues associated with HIV substrate specificity, which previous studies have shown are not the major residues involved in resistance to current drugs. In addition, the ex vivo evolution of HIV PR in the presence and absence of PI will be assessed and information gained will inform the FIV studies. The Aims are to: 1) Prepare a battery of infectious FIV encoding "HIVinized" PR molecules in which multiple residues of FIV PR have been substituted with the equivalent residue of HIV PR. Phenotypic, genotypic, and structural analyses will be performed on virus progeny and substrate specificity changes using an ex vivo Gag processing assay will be assessed;2) Continue to generate a panel of PR resistance mutants using novel PR inhibitors. Ex vivo competition experiments between wild type and drug-resistant HIVs will be carried out as a measure of relative viral fitness. In turn, these HIV PRs will identify additional residues to alter in FIV PR for chimeric PR studies in Aim 1. Lastly, structural evaluation of selected FIV and HIV drug-resistant PRs will be undertaken in order to assess loss of protease inhibitor (PI) potency and altered enzyme function of fully resistant PRs. Findings from these studies will provide insights into the functional plasticity of PR in regard to residue changes leading to resistance and alteration of viral function. PAGE